Project Summary Melanoma is the deadliest form of skin cancer and its incidence is rising rapidly. Our long-term objective is to determine the mechanisms leading to aberrant growth of human melanomas. Mutations in the serine/threonine kinase, B-RAF, are found in approximately 50-70% of melanomas and mutant B-RAF is required for proliferation and tumorigenicity of melanomas. Mutant B-RAF signaling in human melanoma cells elevates activation of the MEK-ERK1/2 pathway and regulates two key G1 cell cycle proteins: cyclin D1 and p27Kip1. Our preliminary data indicate that mutant B-RAF signaling and cyclin D1 regulate p27Kip1 via expression of Skp2 F- box protein, a component of a Skp1/Cullin/F-box (SCF) ubiquitin ligase complex that mediates p27KiP1 degradation. Specifically, mutant B-RAF and cyclin D1 regulate the expression of Cks1, a co-factor for Skp2 that regulates Skp2 protein stability. B-RAF mutations are also found in common acquired and dysplastic nevi melanocytes and sustained B-RAF signaling mediates a cell cycle arrest concomitant with down-regulation of ERK1/2 activation observed in nevi. MKP3, a phosphatase that inactivates ERK1/2, is proteasomally down- regulated in melanoma cells. In this proposal, we seek to further elucidate cell cycle control in melanoma and melanocytes by studying mutant B-RAF-regulated signaling events. Aim 1 will analyze Cks1 levels in human tissue samples, B-RAF depleted melanoma cells in a 3-D skin reconstruct model, and following modulation of E2F transcription factors. Aim 2 will determine the mechanism underlying Skp2 effects on melanoma cell cycle progression in 3-D and 2-D systems. In Aim 3, we will determine whether MKP3 inhibits mutant B-RAF signaling in melanocytes and seek to identify the E3 ubiquitin ligase that regulates MKP3 protein turnover. The lack of effective drugs and treatments for melanoma underscores the need to identify new targets in melanoma. At the completion of our experiments, we aim to have identified new targets for therapeutic intervention to prevent aberrant melanoma cell proliferation.